During the past year, in collaboration with Dr. Richard Childs of NHLBI, we have begun studying T cell alloreactivity in MHC-matched donor-recipient pairs before stem cell allotransplantation, and in recipients post-transplant. To this end we have adapted ELISPOT methods to enumerate the number of alloreactive cells and to characterize their pattern of cytokine production. Initially, considerable time was spent improving the laboratory methods for cryopreservation and thawing samples, and in developing criteria for evaluating the quality of thawed products. One major concern in comparing immune T cell responses to donor and recipient stimulator cells is the marked heterogeneity in composition and viability of thawed mixed mononuclear cell preparations. To address this problem, we have adapted published methods using IL-4 and CD40L which permit the in vitro generation large numbers of homogenous activated B cells from small numbers of mixed mononuclear cells. In comparing the function of these activated B cells with mixed mononuclear cells as APC for stimulating alloreactivity we find that activated B cells are at least as effective as mixed mononuclear cells in eliciting alloreactive T cell proliferation. The nature of the alloimmune response however is quite different. Mixed mononuclear cells typically evoke effectors with a strong Th1/Tc1 pattern of cytokine production. By contrast B cell APC powerfully polarize responding T cells in a Th2/Tc2 pattern of cytokine release. We are currently extending these studies by comparing the impact of B cell and mixed mononuclear cell APC on T cell responses against viral and ultimately against tumor related antigen epitopes as well. Since these patterns of cytokine production would be expected to have significant function consequences, these studies have important implications concerning the use of prepared B cells as APC in a range of other ex vivo immunologic applications. In addition to these differences in cytokine polarization, B and mixed mononuclear cell APC also appear to elicit quantitatively different patterns of T cell response, perhaps reflecting differences in minor transplantation antigen expression. We are still in the process of analyzing the results of our initial studies using these differing populations of APC to study alloreactivity before and after transplantation in Dr. Child's patients. We hope in the near future to determine whether the methods we are developing can be used in predicting or monitoring GVHD or engraftment problems post transplant.